Various methods are presently used for the detection and/or quantitative determination of specific binding agents and/or their corresponding bindable substances. Although these methods differ greatly from each other in sensitivity, ease of operation and chemical and physical principles involved, important similarities are generally recognized. Mostly, the relationship between a specific binding agent and its corresponding bindable substance(s) will be of the acceptor-ligand type, such as, for example, antigen-antibody and receptor-ligand interactions. Antigen-antibody or immunological interactions are by far the most important in this connection, and, particularly for diagnostic purposes, detection methods based on such interactions are the most widely used today.
Various techniques can be employed to detect and optionally quantify the complexes formed between the specific binding agents and bindable substances involved. In certain instances, the complexation reaction will lead to a directly visible signal as a result of agglutination and/or precipitation of the complex itself. This will however not always be the case and, in general, the concentration of binding protein and bindable substance needed to produce such result will be far above the practical and useful limits. In order to circumvent this lack of sensitivity or to detect otherwise un-detectable complexes, various methods have been developed such as, for example, immunodiffusion, immuno-electrophoresis, complement fixation, passive haemagglutination, radio-immuno assay (RIA), immuno-fluorescence, enzyme-linked immuno sorbent assay (ELISA). In the last three methods, the detection of the complex is facilitated by labelling the complex with an easily detectable marker which is either bound directly to the specific binding protein, to a secondary binding protein for which the primary binding protein acts as a bindable substance, or to the bindable substance. In the three methods listed, the marker is respectively a radioactive atom or group, a fluorescent substance or an enzyme. Such methods are described i.a. in Weir's Handbook of Experimental Immunology (1967), Blackwell Scientific Publications, Oxford and Edinburgh and U.S. Pat. No. 3,654,090 (ELISA).
More recently, methods have been developed wherein the complexes formed between specific binding protein and bindable substance are visualized by labelling the said complexes directly or indirectly with colloidal metal particles, particularly gold particles. Depending on the circumstances, these particles can be dectected e.g. by direct visual examination, by microscopic or spectrofotometric techniques. A description of the "sol particle immuno assay" (SPIA) technique and of specific applications and improvements thereof will be found e.g. in U.S. Pat. Nos. 4,313,734, 4,446,238 and 4,420,558.
Use has also been made of a latex agglutination method as described e.g. in U.S. Pat. No. 3,857,931 which has certain advantages over the haemagglutination method mentioned above. Indeed, the red blood cell carriers are themselves antigenic and often cause specific agglutination which interferes with the desired antigen-antibody reaction and renders the efficiency of the determination to a large extent dependent on the nature and composition of the material under analysis. The synthetic polymer latexes employed are devoid of this disadvantage. However, their sensitivity is in general insufficient.
The method according to the present invention differs from the previous methods essentially by the fact that as a marker to detect or determine the complex formed between a specific binding protein and the corresponding bindable substances there are used latex particles which can be detected visually due to their capability to absorb light in the visual spectrum, or to emit light after irradiation. The former detection is based on colour, the latter on fluorescence.